The long-term objective is to determine the mechanisms by which ionizing radiation, through direct effects, alters DNA structure. Direct effects accounts for 30-50% of the in vivo DNA damage for low LET (linear energy transfer) radiations. At high LET, direct effects account for approximately 80% of the damage. Knowledge of the mechanism by which ionizing radiation produces damage in DNA is critical in determining the risks, such as induction of cancer or leukemia, due to low dose and low dose rates of radiation. Achievement of the stated objective will be a major benefit in risk assessment and disease treatment. The specific aims are 1) to improve our understanding of hole and excess-electron trapping and detrapping by DNA, 2) to determine the reactions initiated by detrapping the hole/electron and terminated by formation of thermally stable radicals, 3) to correlate stable lesions with their free radical precursors, and 4) to determine the yields of multiply damaged sites. The approach is to use electron paramagnetic resonance (EPR) spectroscopy to study free radical intermediates formed in oligodeoxynucleotides and DNA. Oligodeoxynucleotides will be studied in crystalline form and plasmid DNA will be studied in the form of films. EPR measurements will be made at 4K to maximize detection sensitivity. X-irradiation (70 kV) will be delivered at 4K, 120K, 240K, RT in order to ascertain the effect of radiation temperature. Stable end products will be analyzed using the same samples as employed for EPR. Strand breaks will be identified using high performance liquid chromatography. Base damage will be determined using gas chromatography/mass spectrometry. Central to our design is the use of DNA samples that are structurally well defined. By employing crystals of known structure, we maximize our knowledge of the relevant sample parameters: base sequence, DNA conformation, hydration state, counter ions, packing, and purity. These experiments will determine which free radical intermediates lead to specific types of stable end products in DNA damaged directly by low LET radiation and reveal how the progression of damage is affected by base sequence, conformation, and packing.